In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with specifically a methyl group, rather than a larger carbon chain, replacing a hydrogen atom. These terms are commonly used in chemistry, biochemistry, soil science, and the biological sciences.
In biological systems, methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA metabolism. Methylation of heavy metals can also occur outside of biological systems. Chemical methylation of tissue samples is also one method for reducing certain histological staining artefacts.
DNA methylation in vertebrates typically occurs at CpG sites (cytosine-phosphate-guanine sites; that is, where a cytosine is directly followed by a guanine in the DNA sequence); this methylation results in the conversion of the cytosine to 5-methylcytosine. The formation of Me-CpG is catalyzed by the enzyme DNA methyltransferase. The bulk of mammalian DNA has about 40% of CpG sites methylated but there are certain areas, known as CpG islands which are GC rich (made up of about 65% CG residues) where none are methylated. These are associated with the promoters of 56% of mammalian genes, including all ubiquitously expressed genes. 1-2% of the human genome are CpG clusters and there is an inverse relationship between CpG methylation and transcriptional activity.
DNA methylation involves the addition of a methyl group to the 5 position of cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring (cytosine and adenine are two of the four bases of DNA). This modification can be inherited through cell division. DNA methylation is typically removed during zygote formation and re-established through successive cell divisions during development. DNA methylation is a crucial part of normal organism development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can “remember where they have been”; in other words, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets. In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements which have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer.
DNA methylation involves the addition of a methyl group to DNA—for example, to the number 5 carbon of the cytosine pyrimidine ring—in this case with the specific effect of reducing gene expression. In adult somatic tissues, DNA methylation typically occurs in a CpG dinucleotide context; non-CpG methylation is prevalent in embryonic stem cells. Bisulfite sequencing is the use of bisulfite treatment of DNA to determine its pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. It is also implicated in repression of transcriptional activity.
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information. The objective of this analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymines) resulting from bisulfite conversion.
Sequencing can be done by pyrosequencing, which differs from Sanger sequencing, relying on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.
The Illumina Methylation Assay using the Infinium II platform uses “BeadChip” technology to generate a comprehensive genome wide profiling of human DNA methylation, similar to bisulfite sequencing and pyrosequencing. According to Staaf et al. (2008), “the Infinium II assay seemed to have dye intensity biases between the two channels used in fluorescence detection. Furthermore, this bias was not eliminated even after the data had gone through normalization algorithms used in the BeadStudio software”.
The samples used for the analysis of DNA methylation biomarkers usually contain high concentrations of background DNA from the tumour. However, tumour-derived DNA is difficult to be detected because it is often present in very low concentrations and can be contaminated substantially with DNA from healthy cells. Thus, methods with sensitive detection capabilities of single copies of methylated DNA in a high amount of unmethylated background DNA are often needed to identify aberrantly methylated tumour-derived DNA in body fluids.
The combination of different types of pre-treatment of sample DNA followed by different analytical steps has resulted in a plethora of techniques for determining DNA methylation patterns and profiles.
In particular, the methods of methylome analysis are divided into 3 groups: restriction enzyme based, Chromatin immunoprecipitation based (ChIP) or affinity based and bisulfite conversion (gene based). Restriction enzyme based methods use methylation-sensitive restriction enzymes for small/large scale DNA methylation analysis by combining the use of methylation-sensitive restriction enzymes with experimental approaches (RLGS, DMH etc.) for global methylation analysis, applied to any genome without knowing the DNA sequence. However, large amounts of genomic DNA are required, making the method unsuitable for the analysis of samples when small amount of DNA is recovered. On the other hand, ChIP based methods are useful for the identification of differential methylated regions in tumours through the precipitation of a protein antigen out of a solution by using an antibody directed against the protein. These methods are protein based, applied extensively in cancer research.
Despite several advantages, protein based methods are limited in detecting methyl (CH3) groups in defined sites, with limitations on the data obtained by the frequency of the restriction enzyme recognition sequence, becoming complex when extra amplification is needed after the antibodies attachment.
MSP is known for its high analytical sensitivity, which however can be influenced by the primer design and the number of PCR cycles. Thus, there is a risk of false-positive results arising, which is claimed to be one of the most significant problems when using the methylation technology in cancer early recognition, so increasing the specificity of methylation detection represents an important step in the development of adequate early recognition tests.
The present inventor has appreciated that existing methods have high cost requiring means to detect the fluorescence and are generally not compatible with standard high-volume manufacturing techniques like CMOS processes.